Bacillus safensis and application thereof

Abstract

The application discloses Bacillus safensis and application thereof, and belongs to the technical field of microorganisms. The application provides Bacillus safensis, and the strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 27870. The Bacillus safensis provided by the application has a significant inhibiting effect on peanut or tobacco rhizome disease pathogenic bacteria such as Sclerotium rolfsii, Cylindrocarpon destructans, Rhizoctonia solani and Sclerotinia homeocarpa, and thus can be applied to the prevention and treatment of peanut or tobacco rhizome disease pathogenic bacteria, so that the yield and quality of peanuts and tobaccos are improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of Bacillus safranin and its applications. Background Technology

[0002] Peanuts and tobacco are important oilseed and cash crops, playing a crucial role in ensuring oil security and increasing farmers' income. In recent years, due to climate warming, crop variety promotion, and changes in farming systems, especially the promotion of dense planting, significant changes have occurred in the field microclimate and soil microecology. The occurrence of root and stem diseases in peanuts and tobacco has become increasingly serious. For example, diseases such as peanut white mold (Sclerotium rolfsii), peanut black rot (Cylindrocladium parasiticum), tobacco root rot (Fusarium oxysporum), and tobacco sclerotinia (Sclerotinia asclerotiorum) are causing increasingly severe damage. Because of the large number of pathogens and significant regional differences, there are currently no effective control methods for root and stem diseases in production, making them the most important diseases affecting crop production.

[0003] Currently, the control of crop diseases and pests mainly relies on resistant plant varieties and chemical control. However, the problem of pest resistance is becoming increasingly serious, and chemical pesticide residues cause serious harm, such as toxicity and environmental pollution, posing a significant threat to human health and the ecosystem, and hindering the sustainable development of industries such as peanuts and tobacco. Therefore, finding an economical, safe, and effective control measure is urgently needed. Biological control measures utilize beneficial microorganisms to kill or reduce the number of pathogens to control the occurrence and development of plant diseases. These beneficial microorganisms are also known as antagonistic microorganisms or biocontrol bacteria. This invention will lay a more research foundation and provide more candidate materials for the biological control of peanut and tobacco diseases and pests. Summary of the Invention

[0004] This invention provides a strain of Bacillus safensis, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27870.

[0005] The aforementioned Bacillus sabina exhibits significant inhibitory effects against Sclerotium rolfsii (peanut white rot fungus), Cylindrocladium parasiticum (peanut black rot fungus), Fusarium oxysporum (tobacco root rot fungus), and Sclerotinia sclerotiorum (tobacco sclerotiorum).

[0006] This invention provides the application of the above-mentioned Bacillus sabensis in the prevention and control of pathogens causing diseases of peanut or tobacco roots and stems.

[0007] In this invention, the pathogens causing root and stem diseases are selected from peanut white mold fungus, peanut black rot fungus, tobacco root rot fungus, or tobacco sclerotium fungus.

[0008] This invention provides the application of the above-mentioned Bacillus safranin in the preparation of formulations for controlling pathogens causing diseases of peanut or tobacco roots and stems.

[0009] This invention provides a microbial preparation containing the aforementioned Bacillus safranin.

[0010] The microbial preparation can be selected from solid or liquid formulations. If it is a liquid formulation, the concentration of Bacillus salsa is preferably greater than 1 × 10⁻⁶. 6 cfu / mL.

[0011] A method for controlling root and stem diseases of peanuts or tobacco includes the following steps: irrigating the roots of peanuts or tobacco with a suspension of *Bacillus salsa* to achieve the purpose of controlling root and stem diseases of peanuts or tobacco; the concentration of the *Bacillus salsa* suspension is greater than 1 × 10⁻⁶. 6 cfu / mL.

[0012] The beneficial effects of this invention are as follows:

[0013] This invention provides a Bacillus safortifolius strain that has a significant inhibitory effect on pathogens causing diseases of peanut or tobacco roots and stems, such as peanut white mold, peanut black rot, tobacco root rot, and tobacco sclerotinia. Therefore, it can be applied to the prevention and control of pathogens causing diseases of peanut or tobacco roots and stems, thereby improving the yield and quality of peanuts and tobacco. Attached Figure Description

[0014] Figure 1 The colony morphology of strain YN-LJG-4;

[0015] Figure 2 Electrophoresis image of 16S rDNA of strain YN-LJG-4;

[0016] Figure 3 An phylogenetic tree was constructed for strain YN-LJG-4 and other bacteria;

[0017] Figure 4 The inhibitory effect of strain YN-LJG-4 on peanut white rot fungus;

[0018] Figure 5 The inhibitory effect of strain YN-LJG-4 on peanut black rot pathogen;

[0019] Figure 6The inhibitory effect of strain YN-LJG-4 on tobacco root rot pathogens;

[0020] Figure 7 The inhibitory effect of strain YN-LJG-4 on Sclerotinia sclerotiorum var. tobaccois was investigated. Detailed Implementation

[0021] Strain isolation and identification:

[0022] The experimental soil was obtained from peanut rhizosphere soil from Lijiang, Yunnan. 5g of rhizosphere soil was placed in an Erlenmeyer flask containing 45mL of sterile water, mixed thoroughly, and then gradually increased in increments of 10... -1 ~10 -6 Dilute the soil suspension. Using a micropipette, pipette 50 μL of soil suspension at different dilutions onto LB agar plates, spread evenly, seal the plates, and incubate them upside down in a 28°C incubator for 48 h. Pick a batch of single colonies from the plates, streak them onto LB agar plates, and incubate them upside down in a 28°C incubator for 1–2 days to obtain a single bacterial strain, which is designated YN-LJG-4.

[0023] Following the methods described in Bergey's Manual of Bacterial Identification (8th Edition), strain YN-LJG-4 was identified by morphological and physiological-biochemical characteristics. The specific results are as follows:

[0024] Morphological characteristics: Colonies are irregular in shape, white, flat, smooth, glossy, and viscous, like... Figure 1 As shown. Microscopic observation revealed that the bacteria were Gram-positive, short rod-shaped.

[0025] Biological characteristics: Strain YN-LJG-4 is Gram-positive, catalase-positive, and VP-positive. It can degrade starch and gum, is a fermentative strain, and can utilize sugars such as glucose, galactose, and sucrose. It can also utilize potassium nitrate and ammonium sulfate, and undergoes a methyl red reaction, as shown in Table 1 below.

[0026] Table 1. Physiological and biochemical characteristics of strain YN-LJG-4

[0027] Evaluation items result Evaluation items result Gram staining + Gelatin liquefaction + VP Measurement + Pectin hydrolysis + catalase + Starch hydrolysis + ammonium sulfate + glucose + potassium nitrate + Galactose + Methyl red test + sucrose + Anaerobic growth - Mannitol +

[0028] Note: "+" indicates a positive result; "-" indicates a negative result.

[0029] Genetic identification:

[0030] Three identical colonies were selected for genomic DNA extraction. The extraction method was mainly performed according to the instructions of the TIANGENTIAN amp BACTERia DNA Kit, and the steps are as follows:

[0031] (1) Take 1 mL of bacterial culture medium, centrifuge at 10,000 rpm for 1 min, and aspirate the supernatant as much as possible;

[0032] (2) Add 200 μL of buffer GA to the bacterial pellet and shake until the bacterial pellet is completely suspended;

[0033] (3) Add 4 μL of RNAase (100 mg / mL) solution, shake for 15 seconds, and let stand at room temperature for 5 minutes;

[0034] (4) Add 20 μL of proteinase K solution to the tube and mix well;

[0035] (5) Add 220 μL buffer GB, shake for 15 seconds, place at 70°C for 10 minutes, and centrifuge to remove water droplets from the inner wall of the tube cap.

[0036] (6) Add 220 μL of anhydrous ethanol and shake thoroughly for 15 seconds. At this time, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0037] (7) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column GB3 (place the adsorption column in the collection tube), centrifuge at 12000 rpm for 30s, discard the waste liquid and place the adsorption column CB3 into the collection tube.

[0038] (8) Add 500 μL of buffer GD to the adsorption column CB3 (check whether anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 30 s, discard the waste liquid and put the adsorption column CB3 into the collection tube.

[0039] (9) Add 700 μL of washing solution PW to the adsorption column CB3 (check whether anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 30 s, discard the waste liquid and put the adsorption column CB3 into the collection tube.

[0040] (10) Add 500 μL of washing solution PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 s, discard the waste liquid and put the adsorption column CB3 into the collection tube.

[0041] (11) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 min, discard the waste liquid, and place the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual rinsing liquid in the adsorption material.

[0042] (12) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μL of elution buffer TE to the middle of the adsorption membrane, place at room temperature for 2-5 min, centrifuge at 12000 rpm for 2 min, collect the solution into a centrifuge tube, and store at -20℃.

[0043] PCR and sequencing:

[0044] (1) The extracted DNA was amplified by PCR according to the instructions of the TaKaRa 16S rDNA Bacterial Identification PCR Kit. The forward primer is shown in SEQ ID NO:1 below, and the reverse primer is shown in SEQ ID NO:2 below. The PCR amplification conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min; 53℃ annealing for 1 min; 72℃ extension for 90 s; 72℃ final extension for 5 min, for a total of 30 cycles. The PCR products were separated by 1% agarose gel electrophoresis, stained with EB, and observed under a 3UV™ Transilluminator (UVP, USA). Electrophoresis showed that the 16S rDNA fragment of strain YN-LJG-4 was approximately 1500 bp in size, which was consistent with the expected design of the kit. The amplification results are shown in […]. Figure 2 .

[0045] Forward primer sequence: 5'-AGAGTTTGATCATGGCTCAG-3' (SEQ ID NO:1)

[0046] Reverse primer sequence: 5'-TTCAGCATTGTTCCATTCGC-3' (SEQ ID NO:2)

[0047] (2) Based on the pre-designed primers and the expected amplified fragment size, and using markers, the target fragment was cut under UV light and the DNA was recovered using the Silica Bead DNA Gel Extraction Kit. The 16S rDNA sequence was determined by Qingdao Qingke Biotechnology Co., Ltd. The sequence results showed that the 16S rDNA fragment of strain YN-LJG-4 consisted of 1504 nt base pairs, and its sequence is shown in SEQ ID NO:3.

[0048] 16S rDNA gene:

[0049]

[0050] The above sequences were compared with those in GenBank (NCBI, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) using the BLAST program. Then, 16S rDNA sequences from species and genera closely related to the test strain were obtained from GenBank for identification. The results showed that the 16S rDNA sequence of strain YN-LJG-4 was highly homologous (100%) to the 16S rDNA sequence of *Bacillus safensis* in the GenBank gene bank. A phylogenetic tree was constructed, as follows... Figure 3 As shown, homology analysis revealed that strain YN-LJG-4 and *Bacillus safensis* (Accession No: KX530779.1) form a separate clade and are the closest in evolutionary distance, reflecting their close phylogenetic relationship. Analysis using DANMAN software showed 100% homology between the two. Combining traditional physiological and biochemical characterization with 16S rDNA sequence analysis, strain YN-LJG-4 was ultimately identified as *Bacillus safensis*.

[0051] This strain was deposited on July 13, 2023, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 27870.

[0052] Other materials used in this invention, unless otherwise stated, are commercially available. Other terms used in this invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and not intended to limit the scope of the invention in any way.

[0053] Example 1

[0054] Antimicrobial inhibition test of pathogens causing root and stem diseases:

[0055] Single colonies of YN-LJG-4 were picked and inoculated into LB medium, and cultured at 28°C and 200 rpm for 12 h. 100 μL of the culture was then transferred to 20 mL of LB medium and cultured in a shaker at 28°C and 180 rpm until the bacterial suspension concentration reached 10⁻⁶. 6cfu / mL. In a sterile operating table, inoculate 0.5 cm diameter mycelial cakes of root and stem disease pathogens onto PDA agar plates. Add 6 μL of YN-LJG-4 culture medium at a distance of 2.5 cm from the mycelial cake, and incubate upside down at 28℃ for 10-15 days. The root and stem disease pathogens were selected from *Sclerotium rolfsii* (peanut white rot fungus), *Cylindrocladium parasiticum* (peanut black rot fungus), *Fusarium oxysporum* (tobacco root rot fungus), and *Sclerotinia sclerotiorum* (tobacco sclerotinia rot). All of these pathogens were isolated, identified, and preserved in our laboratory.

[0056] Inhibition rate (%) = [(Control colony diameter - Treated colony diameter) / Control colony diameter] × 100

[0057] The test results are as follows Figures 4-7 As shown:

[0058] Therefore, strain YN-LJG-4 exhibits an inhibition rate of over 50% against all the aforementioned root and stem disease pathogens, demonstrating a significant antibacterial effect. Consequently, in practical applications, strain YN-LJG-4 can be used to control these root and stem disease pathogens, achieving the goal of inhibiting pathogens and promoting yield.

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A type of Bacillus safranin ( Bacillus safensis ), characterized in that, The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27870.

2. The application of Bacillus sabovellatus as described in claim 1 in the control of pathogens causing diseases of peanut or tobacco roots and stems; The pathogens causing diseases of peanuts or tobacco roots and stems are selected from peanut white mold fungus, peanut black rot fungus, tobacco root rot fungus, or tobacco sclerotium fungus.

3. The use of Bacillus safranin as described in claim 1 in the preparation of formulations for controlling pathogens causing root and stem diseases of peanuts or tobacco; The pathogens causing diseases of peanuts or tobacco roots and stems are selected from peanut white mold fungus, peanut black rot fungus, tobacco root rot fungus, or tobacco sclerotium fungus.

4. A microbial preparation, characterized in that, Contains the Bacillus safortiformis as described in claim 1.

5. The microbial preparation according to claim 4, characterized in that, The microbial preparation is selected from solid or liquid preparations.

6. The microbial preparation according to claim 5, characterized in that, The concentration of Bacillus salsa in the liquid formulation is greater than 1×10⁻⁶. 6 cfu / mL.

7. A method for controlling root and stem pathogens of peanuts or tobacco, characterized in that, The steps are as follows: The bacterial suspension of *Bacillus salsa* as described in claim 1 is applied to the roots of peanuts or tobacco to achieve the purpose of controlling root and stem diseases of peanuts or tobacco; the concentration of the *Bacillus salsa* bacterial suspension is greater than 1 × 10⁻⁶. 6 cfu / mL; The pathogens causing diseases of peanuts or tobacco roots and stems are selected from peanut white mold fungus, peanut black rot fungus, tobacco root rot fungus, or tobacco sclerotium fungus.

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